High-resolution Separation Research Articles

Optimization of an IgG1 CIEF separation by using narrow-range ampholytes and DMSO as protein solubilizer.

CIEF is a powerful separation tool utilized in the characterization and relative quantitation of therapeutic mAb charged isoforms. However, one CIEF method is not capable of separating all mAbs with high resolution and reproducibility. Optimization of sample composition and separation parameters is expected when developing a CIEF method for a specific mAb. This paper summarizes a root cause investigation into why a validated CIEF separation method for MAK33 (a type of IgG1) was no longer reproducible. In addition, this paper introduces the concept of sample focusing volume, which is defined as the actual capillary volume occupied by the sample after focusing and explains why there is less protein precipitation and aggregation when using narrow-range ampholytes than broad-range ampholytes. The use of DMSO as protein solubilizer and possible replacement of urea is also explored in this work. Finally, this paper demonstrates that a new optimized CIEF method can achieve over 100 reproducible high-resolution separations of MAK33 per neutral-coated capillary.

Morphology optimization and assessment of the performance limits of high-porosity nanostructured polymer monolithic capillary columns for proteomics analysis

This study targets the synthesis of high external-porosity poly(styrene-co-divinylbenzene) monolithic support structures with macropore and globule sizes in the sub-micron range, aiming at the realization of high-speed and high-resolution gradient separations of intact proteins and peptides. The thermodynamic and kinetic aspects of the free-radical polymerization synthesis were adjusted by tuning the porogen to monomer ratio, the porogen ratio, the initiator content, and polymerization temperature. Next, column morphology was linked to eddy-dispersion and mobile-phase mass-transfer contributions and the chromatographic performance limits were benchmarked against conventional packed columns and silica monoliths. Polymer monolithic structures yielding a separation impedance as low as 976 were created allowing to generate N > 1,000,000 (for an unretained marker), albeit the expense of very long analysis times. Decreasing the macropore and globule sizes below a certain threshold led to significant increase in eddy dispersion, as globular entities agglomerate, and a small number of large flow-through pores permeate the overall fine interconnected polymer network with small diameter flow-through pores. The potential of monolith chromatography for proteomics application is demonstrated with a ballistic 6 s gradient separation of intact proteins and a high-resolution nanoLC-Orbitrap mass spectrometric analysis of a tryptic E. coli digest applying a coupled-column system.

IN SILICO DEVELOPMENT OF HIGH-RESOLUTION MLVA TYPING SCHEME FOR ENTEROCOCCUS FAECIUM

Emergence of enterococci as nosocomial pathogens is frequently associated with hospital outbreaks. Vancomycin resistance is especially perturbing as it limits the possible therapeutic options. Based on vast in silico analysis, we introduce a new multiple-locus variable-number tandem repeat analysis (MLVA) scheme for genotyping of Enterococcus faecium isolates, parameters of which are comparable to these of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The scheme was tested in silico on all available at this time E. faecium genomes in NCBI Genbank. Searching for suitable variable number of tandem repeats (VNTR) loci was conducted with a set of free access applications. Ten VNTR loci were selected according to their polymorphic structure and stability using the Primer-BLAST utility of NCBI. Primers were designed to be compatible in a multiplex reaction and the method was adapted for high resolution separation techniques. As a result, a total of 60 MLVA profiles and 35 MLST profiles were generated from the analysis of 114 sequenced genomes. Minimum spanning trees were created for both MLVA and MLST in order to analyze the genetic relatedness between isolates. Hunter Gaston discriminatory index was measured for both MLVA (0,959) and MLST (0,926). Typeability was also measured for both methods (MLVA – 85.9%; MLST – 89.4%). These results suggest that the new MLVA scheme is suitable for epidemiological studies of hospital-adapted E. faecium isolates.

Ultra-High-Resolution Ion Mobility Separations Over Extended Path Lengths and Mobility Ranges Achieved using a Multilevel Structures for Lossless Ion Manipulations Module.

Over the past few years, structures for lossless ion manipulations (SLIM) have used traveling waves (TWs) to move ions over long serpentine paths that can be further lengthened by routing the ions through multiple passages of the same path. Such SLIM "multipass" separations provide unprecedentedly high ion mobility resolving powers but are ultimately limited in their ion mobility range because of the range of mobilities spanned in a single pass; that is, higher mobility ions ultimately "overtake" and "lap" lower mobility ions that have experienced fewer passes, convoluting their arrival time distribution at the detector. To achieve ultrahigh resolution separations over broader mobility ranges, we have developed a new multilevel SLIM possessing multiple stacked serpentine paths. Ions are transferred between SLIM levels through apertures (or ion escalators) in the SLIM surfaces. The initial multilevel SLIM module incorporates four levels and three interlevel ion escalator passages, providing a total path length of 43.2 m. Using the full path length and helium buffer gas, high resolution separations were achieved for Agilent tuning mixture phosphazene ions over a broad mobility range (K0 ≈ 3.0 to 1.2 cm2/(V*s)). High sensitivity was achieved using "in-SLIM" ion accumulation over an extended trapping region of the first SLIM level. High transmission efficiency of ions over a broad mobility range (e.g., K0 ≈ 3.0 to 1.67 cm2/(V*s)) was achieved, with transmission efficiency rolling off for the lower mobility ions (e.g., K0 ≈ 1.2 cm2/(V*s)). Resolving powers of up to ∼560 were achieved using all four ion levels to separate reverse peptides (SDGRG1+ and GRGDS1+). A complex mixture of phosphopeptides showed similar coverage could be achieved using one or all four SLIM levels, and doubly charged phosphosite isomers not significantly separated using one SLIM level were well resolved when four levels were used. The new multilevel SLIM technology thus enables wider mobility range ultrahigh-resolution ion mobility separations and expands on the ability of SLIM to obtain improved separations of complex mixtures with high sensitivity.

Conceptual design of a novel and compact superconducting recoil separator for radioactive isotopes

The recent development of radioactive beam facilities, such as the HIE-ISOLDE facility at CERN, has significantly expanded the capabilities for investigating the structure of the atomic nucleus and the nuclear interaction. All these studies require an efficient and high-resolution recoil separator for the unambiguous identification of medium and large mass reaction fragments. In order to meet such needs, a versatile recoil separator for radioisotopes based on a compact superconducting (SC) Fixed-Field Alternating-Gradient (FFAG) ring has been designed. It is based on a mini-ring of approximately 3.5 m circumference formed by superconducting combined function magnets with both the bending and focusing/defocusing functions. This machine will be able to operate with light and heavy radioisotopes and separate the reaction fragments from the main beam by radiofrequency gymnastics. It also presents the advantage of significantly reducing the size with respect to standard non-SC recoil separator configurations. In this article, the first design of this FFAG based recoil separator is presented, and first beam dynamics simulation results are discussed.

Dielectrophoresis of proteins: experimental data and evolving theory.

The ability to selectively move and trap proteins is core to their effective use as building blocks and for their characterization. Analytical and preparative strategies for proteins have been pursued and modeled for nearly a hundred years, with great advances and success. Core to all of these studies is the separation, isolation, purification, and concentration of pure homogeneous fractions of a specific protein in solution. Processes to accomplish this useful solution include biphasic equilibrium (chromatographies, extractions), mechanical, bulk property, chemical equilibria, and molecular recognition. Ultimately, the goal of all of these is to physically remove all non-like protein molecules-to the finest detail: all atoms in the full three-dimensional structure being identical down the chemical bond and bulk structure chirality. One strategy which has not been effectively pursued is exploiting the higher order subtle electrical properties of the protein-solvent system. The advent of microfluidic systems has enabled the use of very high electric fields and well-defined gradients such that extremely high resolution separations of protein mixtures are possible. These advances and recognition of these capabilities have caused a re-evaluation of the underlying theoretical models and they were found to be inadequate. New theoretical descriptions are being considered which align more closely to the total forces present and the subtlety of differences between similar proteins. These are focused on the interfacial area between the protein and hydrating solvent molecules, as opposed to the macroscale assumptions of homogeneous solutions and particles. This critical review examines all data which has been published that place proteins in electric field gradients which induce collection of those proteins, demonstrating a force greater than dispersive effects or countering forces. Evolving theoretical constructs are presented and discussed, and a general estimate of future capabilities using the higher order effects and the high fields and precise gradients of microfluidic systems is discussed. Graphical abstract.

Simultaneous Preconcentration and Separation of Native Protein Variants Using Thermal Gel Electrophoresis.

Proteins must maintain proper folding conformations and express the correct post-translational modifications (PTMs) to exhibit appropriate biological activity. However, assessing protein folding and PTMs is difficult because routine polyacrylamide gel electrophoresis (PAGE) methods lack the separation resolution necessary to identify variants of a single protein. Additionally, standard PAGE denatures proteins prior to analysis precluding determinations of folding states or PTMs. To overcome these limitations, a microfluidic thermal gel electrophoresis platform was developed to provide high-sensitivity, high-resolution analyses of native protein variants. A thermally reversible gel was utilized as a separation matrix while in its solid state (30 °C). This thermal gel provided sufficient separation resolution to identify three variants of a fluorescently labeled model protein. To increase detection sensitivity, analyte preconcentration was conducted in parallel with the separation. Continuous analyte enrichment afforded detection limits of 500 fg of protein (250 pM) while simultaneous baseline separation resolution was achieved between variants. The effects of temperature on thermal gel electrophoresis were also characterized. The unique temperature-dependent outcomes illustrated how method performance can be tuned through a thermal dimension. Ultimately, the high detection sensitivity and separation resolution provided by thermal gel electrophoresis enabled rapid screening of native protein variants.

Fabrication and Characterization of Anisotropic Porous Poly(styrene@acrylic acid) Monolith for Enhanced Ability of Heavy Metal Adsorption

Creating anisotropic interconnected macropores is an efficient way to improve the adsorption ability of adsorbents in terms of fast adsorption, high separation resolution, and high flow hydrodynami...

Recent experiments at the JYFLTRAP Penning trap

The JYFLTRAP double Penning trap mass spectrometer at the Ion Guide Isotope Separator On-Line (IGISOL) facility offers excellent possibilities for high-precision mass measurements of radioactive ions. Around 400 atomic masses, including around 50 isomeric states, have been measured since JYFLTRAP became operational. JYFLTRAP has also been used as a high-resolution mass separator for decay spectroscopy experiments as well as an ion counter for fission yield studies. In this contribution, an overview of recent activities at the JYFLTRAP Penning trap is given, with a focus on nuclei discussed in the PLATAN2019 meeting.

Rapid and Simultaneous Characterization of Drug Conjugation in Heavy and Light Chains of a Monoclonal Antibody Revealed by High-Resolution Ion Mobility Separations in SLIM.

Antibody-drug conjugates (ADCs) have recently gained traction in the biomedical community due to their promise for human therapeutics and an alternative to chemotherapy for cancer. Crucial metrics for ADC efficacy, safety, and selectivity are their drug-antibody ratios (DARs). However, DAR characterization (i.e., determining the average number of conjugated drugs on the antibody) through analytical methods remains challenging due to the heterogeneity of drug conjugation as well as the numerous post-translational modifications possible in the monoclonal antibody. Herein, we report on the use of high-resolution ion mobility spectrometry separations in structures for lossless ion manipulations coupled to mass spectrometry (SLIM IMS-MS) for the rapid and simultaneous characterization of the drug load profile (i.e., stoichiometric distribution of the number of conjugated drugs present on the mAb), determination of the weighted average DAR in both the heavy and light chains of a model antibody-drug conjugate, and calculation of the overall DAR of the ADC. After chemical reduction of the ADC and a subsequent 31.5 m SLIM IMS separation, the various drug-bound antibody species could be well resolved for both chains. We also show significantly higher resolution separations were possible for these large ions with SLIM IMS as compared to ones performed on a commercially available (1 m) drift tube IMS-MS platform. We expect high-resolution SLIM IMS separations will augment the existing toolbox for ADC characterization, particularly to enable the rapid optimization of DAR for a given ADC and thus better understand its potential toxicity and potency.

The fluidic resistance of an array of obstacles and a method for improving boundaries in deterministic lateral displacement arrays

Deterministic lateral displacement (DLD) is a microfluidic method of separating particles by size. DLD relies on precise flow patterns to deliver high-resolution particle separation. These patterns determine which particles are displaced laterally, and which follow the flow direction. Prior research has demonstrated that the lateral array boundaries can be designed to improve the uniformity of the critical size and hence separation performance. A DLD device with an invariant critical size throughout is yet unknown. In this work, we propose a 3D design approach. We first represent the flow through the DLD as a 2D lattice of resistors. This is used to determine the relative flow resistances at the boundaries that will deliver the correct flux patterns. We then use the lattice Boltzmann method to simulate fluid flow in a 3D unit cell of the DLD and measure the fluidic resistance for a range or typical dimensions. The results of this work are used to create a new equation for fluidic resistance as a function of post size, post height, and post spacing. We use this equation to determine array geometries that should have the appropriate resistances. We then design and simulate (in COMSOL) complete devices and measure fluid fluxes and first flow-lane widths along the boundaries. We find that the first flow-lane widths are much more uniform than in any devices described previously. This work provides the best method for designing periodic boundaries, and enables narrower, shorter, and higher throughput DLD devices.

Closed-Loop, Axial Temperature Control of Etched Silicon Microcolumn for Tunable Thermal Gradient Gas Chromatography

Combining the resolution of conventional gas chromatography systems with the size factor of microGC systems is important for improving the affordability and portability of high performance gas analysis. Recent work has demonstrated the feasibility of high resolution separation of gases in a benchtopscale short column system by controlling thermal gradients through the column. This work reports a microfabricated thermally controllable gas chromatographic column with a small footprint (approximately 6.25 cm2). The design of the 20 cm column utilizes 21 individually controllable thin film heaters and conduction cooling to produce a desired temperature profile. The reported device is capable of heating and cooling rates exceeding 8000 °C/min and can reach temperatures of 350 °C. The control methods allow for excellent disturbance rejection and precision to within +/− 1 °C. Each length of the column between heaters was demonstrated to be individually controllable and displayed quadratic temperature profiles. This paper focuses on the fabrication process and implementation of the thermal control strategy. [2019–0113]

Characterization of the Volume-based or Number-based Size Distribution for Silica Nanoparticles Using a Unique Combination of Online Dynamic Light Scattering Having a Uni-tau Multi-bit Correlator and High-resolution Centrifugal Field-Flow Fractionation Separator.

This paper presents a study of the size distributions of colloidal nanoparticles using an online dynamic light scattering (DLS) unit with a uni-tau multi-bit correlator (UMC) combined with a centrifugal field-flow fractionation (CF3) separator. Conventionally, the FFF-UV-MALS system utilizing field-flow fractionation (FFF) combined with a UV detector and multi-angle light scattering instrument (MALS) could be used to obtain the particle size distribution of colloidal nanoparticles. Lately, DLS as a technique to measure the size distributions of colloid materials has become prevalent. However, the DLS instrument will practically measure only the large particles in a multi-modal particle mixture. Therefore, the CF3-DLS w/UMC system that was developed consisted of a CF3 unit connected to an online DLS instrument with UMC. The system could measure the volume- or number-based size distribution with highly quantitative and accurate histograms for multi-modal samples. The size distributions were validated with size distributions obtained by images of an atomic force microscope (AFM). Two types of colloidal silica nanoparticles with different distribution widths were used in this study.

Simultaneous determination of cereal monosaccharides, xylo- and arabinoxylo-oligosaccharides and uronic acids using HPAEC-PAD

Xylo- and arabinoxylo-oligosaccharides (XOS and AXOS) are of interest for their prebiotic activity. The production of these oligomers might be accompanied with monosaccharides. The measurement of both oligosaccharides and monosaccharides usually requires two methods. The current work presents an HPAEC-PAD method based on gradient elution of aqueous solvents sodium hydroxide and sodium acetate, in contrast to conventional isocratic elution, for the simultaneous separation of 16 standards of monosaccharides, xylo-oligosaccharides, arabinoxylo-oligosaccharides and uronic acids using CarboPac PA 200 column. The presented method showed a stable baseline and high-resolution separation of the standards. The method showed acceptable accuracy and precision. Limits of Detection and Quantitation (LOD and LOQ) were estimated for all the standards. The method was applied to measure the activity of a commercial endoxylanase on wheat bran; a steady release of xylose monosaccharide was observed. Enzyme action on oligosaccharide standards showed a preference for the larger oligosaccharides.

A flow distribution and collection feature for ensuring scalable uniform flow in a chromatography device

We discuss how the efficiency of a chromatography device could be enhanced by incorporating a new feature which ensures flow uniformity. The overall flow of fluid within the device, which has a cuboid shape, could be visualized as a combination of two z patterns. The device is therefore designated as cuboid z2. The reason for flow uniformity is explained using a simple mathematical model, and also based on computational fluid dynamic (CFD) simulations. The cuboid z2 device outperformed its equivalent conventional cylindrical column in terms of separation efficiency metrics such as the number of theoretical plates, the reduced plate height, and attributes of non-interacting tracer solute peaks. The results discussed in this paper clearly indicate the suitability of the cuboid z2 device for carrying out high-resolution chromatographic separations. The z2 flow enhancing feature would also be broadly applicable to any process which requires uniform flow in three-dimensional porous media. In addition to superior fluidic attributes, the cuboid z2 chromatography device has other advantages such as compactness and scalability.

High-Resolution capillary electrophoresis separation of large RNA under non-aqueous conditions

Large RNAs including messenger RNAs (mRNAs) are promising candidates for development of new drug products and vaccines. Development of high resolution methods for direct analysis of large RNAs, especially for purity in general and size or length in particular, is critical to support new drug development and manufacture. However, resolution based on size or length for large RNAs is limited even by capillary electrophoresis (CE), which is one of the most efficient separation methods for nucleic acids in general. This paper presents a capillary gel electrophoresis (CGE) method for separating large RNA molecules by size or length under strongly denaturing, non-aqueous conditions. We believe that our work constitutes the first time that a gel suitable for CGE prepared with high molecular weight polymers and using only formamide as solvent has been successfully employed to analyze large RNAs on the basis of their size or length with high resolution. With an eye toward application for mRNAs in particular, separation conditions in this work were optimized for RNAs approximately 2000 nucleotides (nt) in length. As compared to a standard CGE method using an aqueous gel, resolution for commercially-available RNA ladder components at 1500 and 2000 nt is increased approximately 6-fold. The impacts of polymer type, molecular weight of the polymer, and polymer concentration on the separation were studied and optimized. Analysis of the results presented here also provides guidance for optimization of separation conditions for RNAs with different sizes as needed for particular applications in the future.

Atomic-level separation of thiolate-protected metal clusters.

Fine metal clusters have attracted much attention from the viewpoints of both basic and applied science for many years because of their unique physical/chemical properties and functions, which differ from those of bulk metals. Among these materials, thiolate (SR)-protected gold clusters (Aun(SR)m clusters) have been the most studied metal clusters since 2000 because of their ease of synthesis and handling. However, in the early 2000s, it was not easy to isolate these metal clusters. Therefore, high-resolution separation methods were explored, and several atomic-level separation methods, including polyacrylamide gel electrophoresis (PAGE), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC), were successively established. These techniques have made it possible to isolate a series of Aun(SR)m clusters, and much knowledge has been obtained on the correlation between the chemical composition and fundamental properties such as the stability, electronic structure, and physical properties of Aun(SR)m clusters. In addition, these high-resolution separation techniques are now also frequently used to evaluate the distribution of the product and to track the reaction process. In this way, high-resolution separation techniques have played an essential role in the study of Aun(SR)m clusters. However, only a few reviews have focused on this work. This review focuses on PAGE, HPLC, and TLC separation techniques, which offer high resolution and repeatability, and summarizes previous studies on the high-resolution separation of Aun(SR)m and related clusters with the purpose of promoting a better understanding of the features and the utility of these techniques.

Status of the SPES Exotic Beam Facility

At Legnaro National Laboratories of INFN is under construction a Rare Isotope Facility called “Selective Production of Exotic Species” (SPES) based on a 35-70 MeV proton cyclotron, able to deliver two beams with a total current up to 0.75 mA, an ISOL fission target station and an existing ALPI superconducting accelerator as a post accelerator (up to 10 MeV/u for A/q=7). The paper will cover notably: the high-resolution mass separator, the CW RFQ (80 MHz, 727 keV/u, with internal bunching), the 1+ low energy transfer line and the injection line from Charge Breeder to ALPI under installation.

High resolution mass separator dipole design studies for SPES project

The purposes of the SPES (Selective Production of Exotic Species) project at INFN laboratory in Legnaro (Italy) is to study nuclei close to the drip lines. Therefore, a High-Resolution Mass Separator (HRMS) must provide full separation of the ions with a resolution 1/20000, to be sensible to the proton-neutron mass difference in the fission products. SPES HRMS consists of: two 90° magnet dipoles, one electrostatic multipole in between them, six electrostatic quadrupoles, two electrostatic hexapoles and two electrostatic triplets before and after the slits on the object and image point. All these components will be installed on a high voltage platform with a maximum operating voltage of -240 kV. Before entering the HRMS, a 40 keV energy beam go through an RFQ Cooler, designed to have an output energy spread of 1 eV. Mass separation within target resolution is the most critical part: dipoles must provide a magnetic field homogeneity of 4 10−5 throughout beam occupancy (half magnet pole surface), at a field intensity of 0.562 T for the reference ion 132 Sn. Therefore, a very accurate dipole design is mandatory. This contribute will show the studies which lead to a possible dipole design.

Separation of Cytidine 5′-Monophosphate and Guanosine 5′-Monophosphate Based on the Hyper-Cross-Linked Resin XDA-1 with pH Gradient Elution

Cytidine 5′-monophosphate (CMP) and guanosine 5′-monophosphate (GMP) were separated based on hyper-cross-linked resins. The adsorption performances of different resins to the two 5′-ribonucleotides were compared. The adsorption equilibria, kinetics, and dynamic separation process of CMP and GMP with pH gradient elution were studied experimentally and theoretically. The nonpolar resin XDA-1 was selected as the best adsorbent for separating CMP and GMP. A pH-dependent Langmuir isotherm model was developed. The model predicted the adsorption isotherms of CMP and GMP at different pH levels satisfactorily. The surface diffusion model can describe the adsorption kinetics curves of CMP and GMP better than the pseudo-first-order rate model and the pseudo-second-order rate model. CMP and GMP could be both separated with a high separation resolution when the loading amount is 0.031 or 0.093 bed volume (BV). A novel pH-dependent film-surface diffusion model was developed and the model predicted the dynamic separation process of CMP and GMP with pH gradient elution satisfactorily.